Oncolytic adenovirus

ABSTRACT

Viral vectors and methods of making such vectors are described that preferentially kill neoplastic but not normal cells, the preferred vector being an adenovirus that has the endogenous promoters in the E1A and/or E4 regions substituted with a tumor specific promoter which is preferably E2F responsive.

This application claims priority from U.S. Provisional Application No.60/165,638, Filed Nov. 15, 1999.

FIELD OF THE INVENTION

This invention relates to adenovirus vectors, and to methods for makingand using such vectors. More particularly, it relates to improvedadenovirus vectors containing mutations and substitutions in thepromoters of the E1A and/or the E4 regions which confer substantialtumor cell specific oncolytic activity.

BACKGROUND

From the early part of this century, viruses have been used to treatcancer. The approach has been two-fold; first, to isolate or generateoncolytic viruses that selectively replicate in and kill neoplasticcells, while sparing normal cells. Investigators initially used wildtype viruses, and this approach met with some, albeit limited success.While oncolysis and slowing of tumor growth occurred with little or nodamage to normal tissue, there was no significant alteration in thecourse of the disease. See, Smith et al., Cancer 9: 1211–1218 (1956),Cassel, W. A. et al., Cancer 18: 863–868 (1965), Webb, H. E. et al.,Lancet 1: 1206–1209 (1966). See, also, Kenney, S and Pagano, J. J. Natl.Cancer Inst., vol. 86, no. 16, p.1185 (1994).

More recently, and because of the reoccurrence of disease associatedwith the limited efficacy of the use of wild type viruses, investigatorshave resorted to using recombinant viruses that can be delivered at highdoses, and that are replication competent in neoplastic but not normalcells. Such viruses are effective oncolytic agents in their own right,and further, can be engineered to carry and express a transgene thatenhances the anti neoplastic activity of the virus. An example of thisclass of viruses is an adenovirus that is mutant in the E1 B region ofthe viral genome. See, U.S. Pat. No. 5,677,178, and Bischoff, J. R., D.H. Kirn, A. Williams, C. Heise, S. Horn, M. Muna, L. Ng, J. A. Nye, A.Sampson-Johannes, A. Fattaey, and F. McCormick. 1996, Science.274:373–6.

It is important to distinguish the use of replication competent viruses,with or without a transgene for treating cancer, from the secondapproach that investigators have used, which is a non-replicating virusthat expresses a transgene. Here the virus is used merely as a vehiclethat delivers a transgene which, directly or indirectly, is responsiblefor killing neoplastic cells. This approach has been, and continues tobe the dominant approach of using viruses to treat cancer. It has,however, met with limited success, and it appears to be less efficaciousthan replicating viruses. Nevertheless, foreign genes have been insertedinto the E1 region (see McGrory, Virology 163: 614–17 (1988)), the E3region (see Hanke, Virology 177: 437–44 (1990) and Bett, J. Virol. 67:5911–21 (1993)) or into the E3 region of an E1 deleted vector.

As mentioned above, to avoid damage to normal tissues resulting from theuse of high dose viral therapy it is preferred that the virus have amutation that facilitates its replication, and hence oncolytic activityin tumor cells, but renders it essentially harmless to normal cells.This approach takes advantage of the observation that many of the cellgrowth regulatory mechanisms that control normal cell growth areinactivated or lost in neoplastic cells, and that these same growthcontrol mechanisms are inactivated by viruses to facilitate viralreplication. Thus, the deletion or inactivation of a viral gene thatinactivates a particular normal cell growth control mechanism willprevent the virus from replicating in normal cells, but such viruseswill replicate in and kill neoplastic cells that lack the particulargrowth control mechanism.

For example, normal dividing cells transiently lack the growth controlmechanism, retinoblastoma tumor suppressor, that is lacking in andassociated with unrestricted growth in certain neoplastic cells. Theloss of retinoblastoma tumor suppressor gene (RB) gene function has beenassociated with the etiology of various types of tumors. The product ofthis tumor suppressor gene, a 105 kilodalton polypeptide called pRB orp105, is a cell-cycle regulatory protein. The pRB polypeptide inhibitscell proliferation by arresting cells at the G₁ phase of the cell cycle.The pRB protein is a major target of several DNA virus oncoproteins,including adenovirus E1a, SV40 large T Ag, and papillomavirus E7. Theseviral proteins bind and inactivate pRB, and the function of inactivatingpRB is important in facilitating viral replication. The pRB proteininteracts with the E2F transcription factor, which is involved in theexpression of the adenovirus E2 gene and several cellular genes, andinhibits the activity of this transcription factor (Bagchi et al. (1991)Cell 65: 1063; Bandara et al. (1991) Nature 351: 494; Chellappan et al.(1992) Proc. Natl. Acad. Sci. (U.S.A.) 89: 4549.

The adenovirus, oncoproteins E1a, disrupts the pRB/E2F complex resultingin activation of E2F. However, neoplastic or normal dividing cellslacking sufficient functional pRB to complex E2F will not require thepresence of a functional oncoprotein, such as E1a, to possesstranscriptionally active E2F. Therefore, it is believed that replicationdeficient adenovirus species which lack the capacity to complex RB butsubstantially retain other essential replicative functions will exhibita replication phenotype in cells which are deficient in RB function(e.g., normal dividing cells, or cells which are homozygous orheterozygous for substantially deleted RB alleles, cells which compriseRB alleles encoding mutant RB proteins which are essentiallynonfunctional, cells which comprise mutations that result in a lack offunction of an RB protein) but will not substantially exhibit areplicative phenotype in non-replicating, non-neoplastic cells. Suchreplication deficient adenovirus species are referred to as E1a-RB⁽⁻⁾replication deficient adenoviruses.

A cell population (such as a mixed cell culture or a human cancerpatient) which comprises a subpopulation of neoplastic cells anddividing normal cells both lacking RB function, and a subpopulation ofnon-dividing, non-neoplastic cells which express essentially normal RBfunction can be contacted under infective conditions (i.e., conditionssuitable for adenoviral infection of the cell population, typicallyphysiological conditions) with a composition comprising an infectiousdosage of a E1a-RB⁽⁻⁾ replication deficient adenovirus. This results inan infection of the cell population with the E1a-RB⁽⁻⁾ replicationdeficient adenovirus. The infection produces preferential expression ofa replication phenotype in a significant fraction of the cellscomprising the subpopulation of neoplastic and dividing normal cellslacking RB function (RB⁻ cell) but does not produce a substantialexpression of a replicative phenotype in the subpopulation ofnon-dividing neoplastic cells having essentially normal RB function. Theexpression of a replication phenotype in an infected RB⁽⁻⁾cell(neoplastic or dividing normal cells) results in the death of the cell,such as by cytopathic effect (CPE), cell lysis, apoptosis, and the like,resulting in a selective ablation of such RB⁽⁻⁾ cells from the cellpopulation. See, U.S. Pat. Nos. 5,801,029 and 5, 972, 706.

Typically, E1a-RB⁽⁻⁾ replication deficient adenovirus constructssuitable for selective killing of RB(−) neoplastic cells comprisemutations (e.g., deletions, substitutions, frameshifts) which inactivatethe ability of an E1a polypeptide to bind RB protein effectively. Suchinactivating mutations typically occur in the E1a CR1 domain (aminoacids 30–85 in Ad5: nucleotide positions 697–790) and/or the CR2 domain(amino acids 120–139 in Ad5, nucleotide positions 920–967), which areinvolved in binding the p105 RB protein and the p107 protein.Preferably, the CR3 domain (spanning amino acids 150–186) remains and isexpressed as a truncated p289R polypeptide and is functional intransactivation of adenoviral early genes. FIG. 1 portrays schematicallythe domain structure of the E1a-289R polypeptide.

In addition to alterations in the E1a region of adenovirus, it would bedesirable to enhance viral specific killing of neoplastic cells thatlack RB function by constructing viruses that have critical replicativefunctions under the control of transcriptionally active E2F. Theadenovirus replication cycle has two phases: an early phase, duringwhich 4 transcription units E1. E2. E3, and E4 are expressed, and a latephase which occurs after the onset of viral DNA synthesis when latetranscripts are expressed primarily from the major late promoter (MLP).The late messages encode most of the virus's structural proteins. Thegene products of E1, E2 and E4 are responsible for transcriptionalactivation, cell transformation, viral DNA replication, as well as otherviral functions, and are necessary for viral growth. See, Halbert, D.N., et al., 1985, J. Virol. 56:250–7.

If the adenoviral regions that are involved in virus replication couldbe brought under the control of E2F via an E2F responsivetranscriptional unit, this would provide an enhanced adenovirus thatselectively kills neoplastic cells that lack RB function, but not normalcells.

By way of background, the following references are presented relating toadenoviral vectors with alterations in regions involved in viralreplication, including the E4 region, and E2F responsive promoters.

WO 98/091563, inventors Branton et al., presents methods andcompositions for using adenoviral E4 proteins for inducing cell death.

Gao, G-P., et al., describe the use of adenoviral vectors with E1 and E4deletions for liver-directed gene therapy. See. J. Virology, December1996, p. 8934–8943.

WO 98/46779 describes certain adenoviral vectors capable of expressing atransgene comprising a modified E4 region but retaining E4orf3.

Yeh, P., et al describe the expression of a minimal E4 functional unitin 293 cells which permit efficient dual trans-complementation ofadenoviral E1 and E4 regions. See, Yeh, P., et al J. Virology, January1996, pages 559–565.

U.S. Pat. No. 5,885,833 describes nucleic acid constructs comprising anactivator sequence, a promoter module, and a structural gene. Thepromoter module comprises a CHR region and a nucleic acid sequence thatbinds a protein of the E2F family.

Wang, Q. et al., in Gene Ther. 2:775–83 (1995) describe a 293 packagingcell line for propagation of recombinant adenovirus vectors that lack E1and/or E4 regions. To avoid the transactivation effects of the E1A geneproduct in parental 293 cells as well as the over expression of the E4genes, the E4 promoter was replaced by a cellular inducible hormone genepromoter, the mouse alpha inhibin promoter. Krougliak and Grahamdescribe the development of cell lines that express adenovirus type 5E1, E4, and pIX genes, and thus are able to complement replication ofadenovirus mutants defective in each of these regions. See, Krougliak,V. and Graham, F., Human Gene Therapy, vol. 6: p. 1575–1586, 1995. Fang,B., et al. in J. Virol. 71:4798–803 (1997) describe an attenuated,replication incompetent, adenoviral vector that has the E4 promoterreplaced with a synthetic GALA/VP16 promoter that facilitates packagingof the adenoviral vector in 293 cells that stably express the GAL4/VP16transactivator. The virus was made replication incompetent by deletionof the E1 region of the virus.

U.S. Pat. No. 5,670,488 describes adenoviral vectors having one or moreof the E4 open reading frames deleted, but retaining sufficient E4sequences to promote virus replication in vitro, and having a DNAsequence of interest operably linked to expression control sequences andinserted into the adenoviral genome.

U.S. Pat. No. 5,882,877 describes adenoviral vectors having the E1, E2,E3 and E4 regions and late genes of the adenovirus genome deleted andadditionally comprising a nucleic acid of interest operably linked toexpression control sequences.

WO 98/13508 describes selectively targeting malignant cells using an E2Fresponsive promoter operably linked to a transgene of interest.

Neuman, E., et al., show that the transcription of the E2F-1 gene isrendered cell cycle dependent by E2F DNA-binding sites within itspromoter. See, Mol Cell Biol. 15:4660 (1995). Neuman, E., et al alsoshow the structure and partial genomic sequence of the human E2F1 gene.See, Gene. 173:163–9 (1996).

Parr, M. J., et al., show that tumor-selective transgene expression invivo is mediated by an E2F-responsive adenoviral vector. See, Nat Med.3:1145–9 (1996). Adams, P. D., and W. G. Kaelin, Jr. showtranscriptional control by E2F. See, Semin Cancer Biol. 6:99–108 (1995).

Hallenbeck, P., et al., describe vectors for tissue-specificreplication. One such vector is adenovirus that is stated to selectivelyreplicate in a target tissue to provide a therapeutic benefit from thevector per se, or from heterologous gene products expressed from thevector. In the former instance a tissue-specific transcriptionalregulatory sequence is operably linked to a coding region of a gene thatis essential for replication of the vector. Several coding regions aredescribed including E1a, E1B, E2 and E4. See, WO 96/17053 and WO96/17053.

Henderson, et al., in U.S. Pat. No. 5,698,443 shows an adenovirus vectorhaving at least one of the genes E1A, E1B or E4 under thetranscriptional control of a prostate cell specific response element.

It should be apparent that viruses offer another means for treatingcancer. Thus, viruses that selectively replicate in, and kill neoplasticcells would be an invaluable weapon in a physician's arsenal in thebattle against cancer.

SUMMARY OF THE INVENTION

The invention described herein provides recombinant adenoviral vectorsand methods for constructing the same, preferably replication competent,adenoviral vectors that substantially and selectively kill neoplasticcells with little or no killing of non neoplastic cells that have atleast one, and preferably two, adenoviral promoter regions that controlthe expression of immediate early genes altered such that certaintranscriptional nucleotide regulatory start sites are removed, orotherwise inactivated, while retaining those sites that are required, orthat substantially facilitate viral replication, and substituting forthe removal of such nucleotide regulatory start sites, a tumor cellspecific transcriptional unit, and optionally a heterologous genesubstituted for a deleted viral gene.

The invention further provides recombinant viral vectors and methods asdescribed above, wherein the adenoviral promoter regions are preferablythe E1a and/or E4.

In another aspect, the invention provides adenoviral vectors thatsubstantially and selectively kill neoplastic cells with little or nokilling of non neoplastic cells that have certain E1a and E4 promotertranscriptional nucleotide start sites removed, or otherwiseinactivated, and substituting therefore a tumor cell specifictranscriptional unit.

In another aspect, the invention provides adenoviral vectors thatsubstantially and selectively kill neoplastic cells with little or nokilling of non neoplastic cells that have at least certain of the E4promoter transcriptional nucleotide start sites removed, or otherwiseinactivated, while retaining those sites that facilitate viralreplication, including certain of the Sp1, ATF, NF1 and NFIII/Oct-1binding sites, and substituting for the E4 promoter nucleotide startsites a tumor cell specific transcriptional unit.

An object of the invention is a description of an adenoviral vector asdescribed above having the E1a and/or the E4 promoter transcriptionalnucleotide start sites removed and substituted therefore a tumor cellspecific transcriptional unit wherein such adenoviral vectors furtherexhibit mutations (e.g. deletions, substitutions, frameshifts) whichinactivate the ability of an E1a polypeptide to bind RB proteineffectively.

A further feature of the invention consists of substituting for the E1aand/or E4 promoter nucleotide start sequences referred to above with atumor cell specific transcriptional unit, one that is responsive to thepRb signaling pathway, including pRb/p107, E2F-1/-2/-3, G1 cyclin/cdkcomplexes, and preferably the promoter is E2F responsive.

The invention also presents methods for preventing or treating disease,and preferably disease resulting from hyperproliferative cell growth,including neoplastic disease using the adenoviral vectors describedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 portrays schematically the domain structure of the E1a-289Rpolypeptide.

FIG. 2 shows the adenoviral E4 promoter.

FIG. 3 shows diagrammatically the invention E4 shuttle vector and theposition of the restriction sites, SpeI (SEQ ID NOS: 26 and 27; SEQ IDNOS 28 and 29) and XhoI (SEQ ID NOS: 30 and 31; SEQ ID NO: 32 and 33),which facilitates substitution of the E4 promoter with a promoter ofchoice.

DETAILED DESCRIPTION OF THE INVENTION

All publications, including patents and patent applications, mentionedin this specification are herein incorporated by reference to the sameextent as if each individual publication was specifically andindividually indicated to be incorporated by reference in its entirety.

Furthermore, it is important to note that while the invention adenoviralvectors' oncolytic activity is ascribed to a mechanism of actioninvolving molecules in the pRb pathway that affect the expression ofviral genes under the control of an E2F responsive promoter, theinvention should not be construed as limited by this mechanism. Ratherit will be appreciated that the invention adenoviral vectors' oncolyticactivity is a function of its structural elements which are thought to,but may not exert oncolysis through the pRb pathway. Thus, the inventionadenoviral vectors derive their tumor versus normal cell killingselectivity by having at least one E2F responsive promoter drivingeither E1a or E4 gene expression. The preferred adenoviral vector is onehaving 2 E2F responsive promoters, one substituted for the E1a promoterand the other for the E4 promoter, as described below.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Generally, the nomenclatureused herein and the laboratory procedures described below are those wellknown and commonly employed in the art.

Standard techniques are used for recombinant nucleic acid methods,polynucleotide synthesis, and microbial culture and transformation(e.g., electroporation, lipofection). Generally, enzymatic reactions andpurification steps are performed according to the manufacturer'sspecifications. The techniques and procedures are generally performedaccording to conventional methods in the art and various generalreferences (see generally, Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd. edition (1989) Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.) which are provided throughout thisdocument. The nomenclature used herein and the laboratory procedures inanalytical chemistry, organic synthetic chemistry, and pharmaceuticalformulation described below are those well known and commonly employedin the art. Standard techniques are used for chemical syntheses,chemical analyses, pharmaceutical formulation and delivery, andtreatment of patients.

Those skilled in the art will also recognize publications thatfacilitate genetic engineering of the invention adenovirus to producethe invention E1A and/or E4 shuttle vectors. Such would include the workof Hitt, M., et al Construction and propagation of human adenovirusvectors. In: Cell Biology: a Laboratory Handbook; J. Celis (Ed),Academic Press, New York (1996); Graham, F. L. and Prevec, L. Adenovirusbased expression vectors and recombinant vaccines. In: Vaccines: NewApproaches to Immunological Problems. R. W. Ellis (ed) Butterworth. Pp.363–390; and Graham, F. L. and Prevec, L. Manipulation of adenovirusvectors. In: Methods in Molecular Biology, Vol. 7: Gene Transfer andExpression Techniques. E. J. Murray and J. M. Walker (eds) Humana PressInc., Clifton, N.J. pp 109–128, 1991. The materials and methodsdescribed in these articles were or could be used below.

In the formulae representing selected specific embodiments of thepresent invention, the amino- and carboxy-terminal groups, althoughoften not specifically shown, will be understood to be in the form theywould assume at physiological pH values, unless otherwise specified. Theamino acid residues described herein are preferably in the “L” isomericform. Stereoisomers (e.g., D-amino acids) of the twenty conventionalamino acids, unnatural amino acids such as a,a-distributed amino acids,N-alkyl amino acids, lactic acid, and other unconventional amino acidsmay also be suitable components for polypeptides of the presentinvention, as long as the desired functional property is retained by thepolypeptide. For the peptides shown, each encoded residue whereappropriate is represented by a three letter designation, correspondingto the trivial name of the conventional amino acid, in keeping withstandard polypeptide nomenclature (described in J. Biol. Chem.,243:3552–59 (1969) and adopted at 37 CFR § 1.822(b)(2)).

As employed throughout the disclosure, the following terms, unlessotherwise indicated, shall be understood to have the following meanings:

The term “inactivated” as applied to “adenoviral transcriptionalnucleotide regulatory site” sequences means rendering such sequences nonfunctional by mutation, including by deletion of all or part of thesequences, or insertion of other sequences into the adenoviraltranscriptional nucleotide sequences thereby rendering them nonfunctional.

The term “adenovirus” as referred to herein indicates over 47 adenoviralsubtypes isolated from humans, and as many from other mammals and birds.See, Strauss, “Adenovirus infections in humans,” in The Adenoviruses,Ginsberg, ed., Plenum Press, New York, N.Y., pp. 451–596 (1984). Theterm preferably applies to two human serotypes, Ad2 and Ad5.

The term “polynucleotide” as referred to herein means a polymeric formof nucleotides of at least 10 bases in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide. Theterm includes single and double stranded forms of DNA.

The term “oligonucleotide” referred to herein includes naturallyoccurring, and modified nucleotides linked together by naturallyoccurring, and non-naturally occurring oligonucleotide linkages.Oligonucleotides are a polynucleotide subset with 200 bases or fewer inlength. Preferably oligonucleotides are 10 to 60 bases in length.Oligonucleotides are usually single stranded, e.g. for probes; althougholigonucleotides may be double stranded, e.g. for use in theconstruction of a gene mutant. Oligonucleotides of the invention can beeither sense or antisense oligonucleotides.

As used herein, the terms “label” or “labeled” refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcolorimetric methods). Various methods of labeling polypeptides andglycoproteins are known in the art and may be used.

By the phrase “tumor cell specific,” as applied to the selectivity ofkilling of the invention adenoviruses, is meant tumor cells that arekilled by the expression of viral genes operably linked to an E2Fresponsive promoter. Considering that E2F is expressed by normal cell,particularly dividing normal cells, it would be expected that theinvention adenoviruses will also kill dividing normal cells, albeit, toa lesser degree than tumor cells.

The term “sequence homology” referred to herein describes the proportionof base matches between two nucleic acid sequences or the proportionamino acid matches between two amino acid sequences. When sequencehomology is expressed as a percentage, e.g., 50%, the percentagedenotes, the proportion of matches over the length of sequence that iscompared to some other sequence. Gaps (in either of the two sequences)are permitted to maximize matching; gap lengths of 15 bases or less areusually used, 6 bases or less are preferred with 2 bases or less morepreferred.

The term “corresponds to” is used herein to mean that a polynucleotidesequence is homologous (i.e., is identical, not strictly evolutionarilyrelated) to all or a portion of a reference polynucleotide sequence, orthat a polypeptide sequence is identical to a reference polypeptidesequence. In contradistinction, the term “complementary to” is usedherein to mean that the complementary sequence is homologous to all or aportion of a reference polynucleotide sequence. For illustration, thenucleotide sequence “TATAC” corresponds to a reference sequence “TATAC”and is complementary to a reference sequence “GTATA”.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides: “reference sequence”, “comparisonwindow”, “sequence identity”, “percentage of sequence identity”, and“substantial identity”. A “reference sequence” is a defined sequenceused as a basis for a sequence comparison; a reference sequence may be asubset of a larger sequence, for example, as a segment of a full-lengthcDNA or gene sequence given in a sequence listing may comprise acomplete cDNA or gene sequence. Generally, a reference sequence is atleast 20 nucleotides in length, frequently at least 25 nucleotides inlength, and often at least 50 nucleotides in length. Since twopolynucleotides may each (1) comprise a sequence (i.e., a portion of thecomplete polynucleotide sequence) that is similar between the twopolynucleotides, and (2) may further comprise a sequence that isdivergent between the two polynucleotides, sequence comparisons betweentwo (or more) polynucleotides are typically performed by comparingsequences of the two polynucleotides over a “comparison window” toidentify and compare local regions of sequence similarity. A “comparisonwindow,” as may be used herein, refers to a conceptual segment of atleast 20 contiguous nucleotide positions wherein a polynucleotidesequence may be compared to a reference sequence of at least 20contiguous nucleotides and wherein the portion of the polynucleotidesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. Optimal alignment of sequences for aligning acomparison window may be conducted by the local homology algorithm ofSmith and Waterman (1981) Adv. Appl. Math. 2: 482, by the homologyalignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988)Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package Release 7.0, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by inspection, and the bestalignment (i.e., resulting in the highest percentage of homology overthe comparison window) generated by the various methods is selected. Theterm “sequence identity” means that two polynucleotide sequences areidentical (i.e., on a nucleotide-by-nucleotide basis) over the window ofcomparison. The term “percentage of sequence identity” is calculated bycomparing two optimally aligned sequences over the window of comparison,determining the number of positions at which the identical nucleic acidbase (e.g., A, T, C, G, U, or I) occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison (i.e., thewindow size), and multiplying the result by 100 to yield the percentageof sequence identity. The terms “substantial identity” as used hereindenotes a characteristic of a polynucleotide sequence, wherein thepolynucleotide comprises a sequence that has at least 85 percentsequence identity, preferably at least 90 to 95 percent sequenceidentity, more usually at least 99 percent sequence identity as comparedto a reference sequence over a comparison window of at least 20nucleotide positions, frequently over a window of at least 25–50nucleotides, wherein the percentage of sequence identity is calculatedby comparing the reference sequence to the polynucleotide sequence whichmay include deletions or additions which total 20 percent or less of thereference sequence over the window of comparison. The reference sequencemay be a subset of a larger sequence.

It is important to note that while a preferred embodiment of theinvention is the incorporation of the human E2F-1 promoter, a promoterthat is “substantially identical” is intended to come within thedefinition of an E2F responsive promoter.

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present. Generally, a substantially purecomposition will comprise more than about 80 percent of allmacromolecular species present in the composition, more preferably morethan about 85%, 90%, 95%, and 99%. Most preferably, the object speciesis purified to essential homogeneity (contaminant species cannot bedetected in the composition by conventional detection methods) whereinthe composition consists essentially of a single macromolecular species.

The term “polypeptide fragment” or “peptide fragment” as used hereinrefers to a polypeptide that has an amino-terminal and/orcarboxy-terminal deletion, but where the remaining amino acid sequenceis identical to the corresponding positions in the naturally-occurringsequence deduced, for example, from a full-length cDNA sequence.Fragments typically 8–10 amino acids long, preferably at least 10–20amino acids long, and even more preferably 20–70 amino acids long.

By the phrase “pRB pathway,” or “pRb signaling pathway” is meant, atleast in part, molecules that affect pRb activity including pRb/p107,E2F-1/-2/-3, and G1 cyclin/cdk complexes. It will be appreciated thatmolecules not presently known may also come within this definition.These molecules mediate their biological effects, at least in part, atthe level of transcription through an E2F responsive promoter.

Other chemistry terms herein are used according to conventional usage inthe art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms(ed. Parker, S., 1985), McGraw-Hill, San Francisco, incorporated hereinby reference.

The production of proteins from cloned genes by genetic engineering iswell known. See, e.g. U.S. Pat. No. 4,761,371 to Bell et al. at column6, line 3 to column 9, line 65. The discussion which follows isaccordingly intended as an overview of this field, and is not intendedto reflect the full state of the art.

DNA which encodes proteins may be inserted into the E1A and/or E4adenoviral constructs of the invention, in view of the instantdisclosure, by chemical synthesis, by screening reverse transcripts ofmRNA from appropriate cells or cell line cultures, by screening genomiclibraries from appropriate cells, or by combinations of theseprocedures, as illustrated below. For example, one embodiment of theinvention is the expression of genes that encode prodrug activityenzymes where such genes are incorporated into regions of the inventionadenoviruses that do not affect their ability to replicate. Screening ofmRNA or genomic DNA may be carried out with oligonucleotide probesgenerated from known gene sequence information. Probes may be labeledwith a detectable group.

In the alternative, a gene sequence may be recovered by use of thepolymerase chain reaction (PCR) procedure. See U.S. Pat. No. 4,683,195to Mullis et al. and U.S. Pat. No. 4,683,202 to Mullis.

A vector is a replicable DNA construct, and is used either to amplifyDNA encoding a desired protein and/or to express DNA which encodes theprotein. An expression vector is a replicable DNA construct in which aDNA sequence encoding a protein of interest is operably linked tosuitable control sequences capable of effecting the expression of theprotein in a suitable host. The need for such control sequences willvary depending upon the host selected and the transformation methodchosen. Generally, control sequences include a transcriptional promoter,an optional operator sequence to control transcription, a sequenceencoding suitable mRNA ribosomal binding sites, and sequences whichcontrol the termination of transcription and translation. Amplificationvectors do not require expression control domains. All that is needed isthe ability to replicate in a host, usually conferred by an origin ofreplication, and a selection gene to facilitate recognition oftransformants.

DNA regions are operably linked when they are functionally related toeach other. For example: a promoter is operably linked to a codingsequence if it controls the transcription of the sequence; a ribosomebinding site is operably linked to a coding sequence if it is positionedso as to permit translation. Generally, operably linked means contiguousand, in the case of leader sequences, contiguous and in reading frame. Apreferred embodiment promoter of the instant invention in thoseinstances where endogenous adenoviral E1a and/or E4 region promotertranscriptional nucleotide regulatory start sites are removed is thesubstitution with a tumor cell specific promoter, one that isresponsive, directly or indirectly, to molecules in the pRb signalingpathway, including the proteins pRb/p107, E2F-1/-2/-3, G1 cyclin/cdkcomplexes, and preferably the promoter is E2F responsive, and morepreferably the promoter is the human E2F-1.

By responsive to molecules in the pRb signaling pathway, is meant thekilling of tumor cells caused by the expression of viral genes under thecontrol an E2F responsive promoter.

Suitable host cells for use in the invention include prokaryotes, yeastcells, or higher eukaryotic cells. Prokaryotes include gram negative orgram positive organisms, for example Eschlerichlia coli (E. coli) orBacilli. Higher eukaryotic cells include established cell lines ofmammalian origin as described below. Exemplary host cells are DH5a, E.coli W3110 (ATCC 27,325), E coli B, E. coli X1776 (ATCC 31,537) and E.coli 294 (ATCC 31,446).

Cultures of cells derived from multicellular organisms are a desirablehost for recombinant protein synthesis. In principal, any highereukaryotic cell culture is workable, whether from vertebrate orinvertebrate culture. However, mammalian cells are preferred.Propagation of such cells in cell culture has become a routineprocedure. See Tissue Culture, Academic Press, Kruse and Paterson,editors (1973). Examples of useful host cell lines are VERO and HeLacells, Chinese hamster ovary (CHO) cell lines, and FL5.12, WI138, BHK,COS-7, CV, and MDCK cell lines. Expression vectors for such cellsordinarily include (if necessary) an origin of replication, a promoterlocated upstream from the gene to be expressed, along with a ribosomebinding site, RNA splice site (if intron-containing genomic DNA isused), a polyadenylation site, and a transcriptional terminationsequence.

As used herein, the term “replication deficient virus” refers to a virusthat preferentially inhibits cell proliferation, causes cell lysis, orinduces apoptosis (collectively considered killing) in a predeterminedcell population (e.g., tumor cells responsive to molecules in the pRbsignaling pathway) which supports expression of a virus replicationphenotype, and which is substantially unable to inhibit cellproliferation, cause cell lysis, induce apoptosis, or express areplication phenotype in non-replicating, non-transformed cells.

The term “RB function” refers to the property of having an essentiallynormal level of a polypeptide encoded by the RB gene (i.e., relative tonon-neoplastic cells of the same histological type), wherein the RBpolypeptide is capable of binding an E1a protein of wild-type adenovirus2 or 5. For example, RB function may be lost by production of aninactive (i.e., mutant) form of RB or by a substantial decrease or totalloss of expression of pRB polypeptide(s), or by an alteration in one ormore of the molecules in the pRb pathway that effect pRb levels.Alternatively, “RB function” refers to the normal transcriptionalactivity of genes, in terms of time of expression and amounts ofproteins expressed, that are under the control of an E2F responsive, pRbpathway sensitive, promoter.

RB function may be substantially absent in neoplastic cells thatcomprise RB alleles encoding a wild-type RB protein; for example, agenetic alteration outside of the RB locus, such as a mutation thatresults in aberrant subcellular processing or localization of RB, or amolecule in the pRB pathway, may result in a loss of RB function.

The term “replication phenotype” refers to one or more of the followingphenotypic characteristics of cells infected with a virus such as areplication deficient adenovirus: (1) substantial expression of lategene products, such as capsid proteins (e.g., adenoviral penton basepolypeptide) or RNA transcripts initiated from viral late genepromoter(s), (2) replication of viral genomes or formation ofreplicative intermediates, (3) assembly of viral capsids or packagedvirion particles, (4) appearance of cytopathic effect (CPE) in theinfected cell, (5) completion of a viral lytic cycle, and (6) otherphenotypic alterations which are typically contingent upon abrogation ofRB function in non-neoplastic cells infected with a wild-typereplication competent DNA virus encoding functional oncoprotein(s). Areplication phenotype comprises at least one of the listed phenotypiccharacteristics, preferably more than one of the phenotypiccharacteristics.

The term “antineoplastic replication deficient virus” is used herein torefer to a recombinant virus which has the functional property ofinhibiting development or progression of a neoplasm in a human, bypreferential cell killing, whether by lysis or apoptosis of infectedneoplastic cells relative to infected non-replicating, non-neoplasticcells of the same histological cell type.

As used herein, “neoplastic cells” and “neoplasia” refer to cells whichexhibit relatively autonomous growth, so that they exhibit an aberrantgrowth phenotype characterized by a significant loss of control of cellproliferation. Neoplastic cells comprise cells which may be activelyreplicating or in a temporary non-replicative resting state (G₁ or G₀);similarly, neoplastic cells may comprise cells which have awell-differentiated phenotype, a poorly-differentiated phenotype, or amixture of both type of cells. Thus, not all neoplastic cells arenecessarily replicating cells at a given timepoint. The set defined asneoplastic cells consists of cells in benign neoplasms and cells inmalignant (or frank) neoplasms. Frankly neoplastic cells are frequentlyreferred to as tumor cells or cancer cells, typically termed carcinomaif originating from cells of endodermal or ectodermal histologicalorigin, or sarcoma if originating from cell types derived from mesoderm.

As used herein, “physiological conditions” refers to an aqueousenvironment having an ionic strength, pH, and temperature substantiallysimilar to conditions in an intact mammalian cell or in a tissue spaceor organ of a living mammal. Typically, physiological conditionscomprise an aqueous solution having about 150 mM NaCl (or optionallyKCl), pH 6.5–8.1, and a temperature of approximately 20–45° C.Generally, physiological conditions are suitable binding conditions forintermolecular association of biological macromolecules. For example,physiological conditions of 150 mM NaCl, pH 7.4, at 37° C. are generallysuitable.

Embodiments of the Invention

The E1a and E4 regions of adenovirus are essential for an efficient andproductive infection of human cells. The E1a gene is the first viralgene to be transcribed in a productive infection, and its transcriptionis not dependent on the action of any other viral gene products.However, the transcription of the remaining early viral genes requiresE1a gene expression. The E1a promoter, in addition to regulating theexpression of the E1a gene, also integrates signals for packaging of theviral genome as well as sites required for the initiation of viral DNAreplication. See, Schmid, S. I., and Hearing, P. in Current Topics inMicrobiology and Immunology, vol. 199: pages 67–80 (1995).

The invention as applied to E1a adenoviral vectors involves thereplacement of the basic adenovirus E1a promoter, including the CAATbox, TATA box and start site for transcription initiation, with a basicpromoter that exhibits tumor specificity, and preferably is E2Fresponsive, and more preferably is the human E2F-1 promoter. Thus, thisvirus will be repressed in cells that lack molecules, or such moleculesare non functional, that activate transcription from the E2F responsivepromoter. Normal non dividing, or quiescent cells, fall in this class,as the transcription factor, E2F, is bound to pRb, or retinoblastomaprotein, thus making E2F unavailable to bind to and activate the E2Fresponsive promoter. In contrast, cells that contain free E2F shouldsupport E2F based transcription. An example of such cells are neoplasticcells that lack pRb function, allowing for a productive viral infectionto occur.

Retention of the enhancer sequences, packaging signals, and DNAreplication start sites which lie in the E1a promoter will ensure thatthe adenovirus infection proceeds to wild type levels in the neoplasticcells that lack pRb function. In essence, the modified E1a promoterconfers tumor specific transcriptional activation resulting insubstantial tumor specific killing, yet provides for enhanced safety innormal cells.

In creating the E1a adenoviral vector by substituting the endogenous E1apromoter with the E2F responsive promoter, the elements upstream ofnucleotide 375 in the adenoviral 5 genome are kept intact. Thenucleotide numbering is as described by See, Schmid, S. I., and Hearing,P. Current Topics in Microbiology and Immunology, vol. 199: pages 67–80(1995). This includes all of the seven A repeat motifs identified forpackaging of the viral genome (See FIG. 2 of Schmid and Hearing, above.)Sequences from nucleotide 375 to nucleotide 536 are deleted by a BsaAIto BsrBI restriction start site, while still retaining 23 base pairsupstream of the translational initiation codon for the E1A protein. AnE2F responsive promoter, preferably human E2F-1 is substituted for thedeleted endogenous E1a promoter sequences using known materials andmethods. The E2F-1 promoter may be isolated as described in Example 1.

The E4 region has been implicated in many of the events that occur latein adenoviral infection, and is required for efficient viral DNAreplication, late mRNA accumulation and protein synthesis, splicing, andthe shutoff of host cell protein synthesis. Adenoviruses that aredeficient for most of the E4 transcription unit are severely replicationdefective and, in general, must be propagated in E4 complementing celllines to achieve high titers. The E4 promoter is positioned near theright end of the viral genome and governs the transcription of multipleopen reading frames (ORF). A number of regulatory elements have beencharacterized in this promoter that are critical for mediating maximaltranscriptional activity. In addition to these sequences, the E4promoter region contains regulatory sequences that are required forviral DNA replication. A depiction of the E4 promoter and the positionof these regulatory sequences can be seen in FIGS. 2 and 3.

Another embodiment of the invention is the generation of an adenoviralvector that has the E4 basic promoter substituted with one that has beendemonstrated to show tumor specificity, preferably an E2F responsivepromoter, and more preferably the human E2F-1 promoter. The reasons forpreferring an E2F responsive promoter to drive E4 expression are thesame as were discussed above in the context of an E1a adenoviral vectorhaving the E1a promoter substituted with an E2F responsive promoter. Thetumor suppressor function of pRb correlates with its ability to repressE2F-responsive promoters such as the E2F-1 promoter (Adams, P. D., andW. G. Kaelin, Jr. 1995, Cancer Biol. 6:99–108; Sellers, W. R., and W. G.Kaelin. 1996, published erratum appears in Biochim Biophys Acta 1996Dec. 9; 1288(3):E-1, Biochim Biophys Acta. 1288:M1–5. Sellers, W. R., J.W. Rodgers, and W. G. Kaelin, Jr. 1995, Proc Natl Acad Sci USA.92:11544–8.) The human E2F-1 promoter has been extensively characterizedand shown to be responsive to the pRb signaling pathway, includingpRb/p107, E2F-1/-2/-3, and G1 cyclin/cdk complexes, and E1A (Johnson, D.G., K. Ohtani, and J. R. Nevins. 1994, Genes Dev. 8:1514–25; Neuman, E.,E. K. Flemington, W. R. Sellers, and W. G. Kaelin, Jr. 1995. Mol CellBiol. 15:4660; Neuman, E., W. R. Sellers, J. A. McNeil, J. B. Lawrence,and W. G. Kaelin, Jr. 1996, Gene. 173:163–9.) Most, if not all, of thisregulation has been attributed to the presence of multiple E2F sitespresent within the E2F-1 promoter. Hence, a virus carrying this (these)modification(s) would be expected to be attenuated in normal cells thatcontain an intact (wild type) pRb pathway, yet exhibit a normalinfection/replication profile in cells that are deficient for pRb'srepressive function. In order to maintain the normalinfection/replication profile of this mutant virus we have retained theinverted terminal repeat (ITR) at the distal end of the E4 promoter asthis contains all of the regulatory elements that are required for viralDNA replication (Hatfield, L. and P. Hearing. 1993, J. Virol. 67:3931–9;Rawlins, D. R., P. J. Rosenfeld, R. J. Wides, M. D. Challberg, and T. J.Kelly, Jr. 1984, Cell. 37:309–19; Rosenfeld, P. J., E. A. O'Neill, R. J.Wides, and T. J. Kelly. 1987, Mol Cell Biol. 7:875–86; Wides, R. J., M.D. Challberg, D. R. Rawlins, and T. J. Kelly. 1987, Mol Cell Biol.7:864–74). This facilitates attaining wild type levels of virus in pRbpathway deficient tumor cells infected with this virus.

In the invention adenoviral constructs involving the E4 region, the E4promoter is preferably positioned near the right end of the viral genomeand it governs the transcription of multiple open reading frames (ORFs)(Freyer, G. A., Y. Katoh, and R. J. Roberts. 1984, Nucleic Acids Res.12:3503–19; Tigges, M. A., and H. J. Raskas. 1984. Splice junctions inadenovirus 2 early region 4 mRNAs: multiple splice sites produce 18 to24 RNAs. J. Virol. 50:106–17; Virtanen, A. P. Gilardi, A. Naslund, J. M.LeMoullec, U. Pettersson, and M. Perricaudet. 1984, J. Virol.51:822–31.) A number of regulatory elements have been characterized inthis promoter that mediate transcriptional activity (Berk, A. J. 1986,Annu Rev Genet. 20:45–79; Gilardi, P., and M. Perricaudet. 1986, NucleicAcids Res. 14:9035–49; Gilardi, P., and M. Perricaudet. 1984, NucleicAcids Res. 12:7877–88; Hanaka, S., T. Nishigaki, P. A. Sharp, and H.Handa. 1987, Mol Cell Biol. 7:2578–87; Jones, C., and K. A. Lee. 1991,Mol Cell Biol. 11:4297–305; Lee, K. A., and M. R. Green. 1987, Embo J.6:1345–53.) In addition to these sequences, the E4 promoter regioncontains elements that are involved in viral DNA replication (Hatfield,L., and P. Hearing. 1993, J Virol. 67:3931–9; Rawlins, D. R., P. J.Rosenfeld, R. J. Wides, M. D. Challberg, and T. J. Kelly, Jr. 1984,Cell. 37:309–19; Rosenfeld, P. J., E. A. O'Neill, R. J. Wides, and T. J.Kelly. 1987, Mol Cell Biol. 7:875–86; Wides, R. J., M. D. Challberg, D.R. Rawlins, and T. J. Kelly. 1987, Mol Cell Biol. 7:864–74.) A depictionof the E4 promoter and the position of these regulatory sequences can beseen in FIGS. 1 and 2. See, also, Jones, C., and K. A. Lee. Mol CellBiol. 11:4297–305 (1991). With these considerations in mind, an E4promoter shuttle was designed by creating two novel restrictionendonuclease sites: a XhoI site at nucleotide 35,576 and a SpeI site atnucleotide 35,815 (see FIG. 3). Digestion with both XhoI and SpeIremoves nucleotides from 35,581 to 35,817. This effectively eliminatesbases −208 to +29 relative to the E4 transcriptional start site,including all of the sequences that have been shown to have maximalinfluence on E4 transcription. In particular, this encompasses the twoinverted repeats of E4F binding sites that have been demonstrated tohave the most significant effect on promoter activation. However, allthree Sp1 binding sites, two of the five ATF binding sites, and both ofthe NF1 and NFIII/Oct-1 binding sites that are critical for viral DNAreplication are retained. Also, many of the E4 promoter elements thatare removed can be substituted with sites that retain similar functions(e.g., transcriptional start site and the TATA box), yet now confertumor cell specificity through the E2F responsive promoter sites.

The preferred E2F responsive promoter is the human E2F-1 promoter. Keyregulatory elements in the E2F-1 promoter that mediate the response tothe pRb pathway have been mapped both in vitro and in vivo (Johnson, D.G., K. Ohtani, and J. R. Nevins. 1994, Genes Dev. 8:1514–25; Neuman, E.,E. K. Flemington, W. R. Sellers, and W. G. Kaelin, Jr. 1995, Mol CellBiol. 15:4660; Parr, M. J., Y. Manome, T. Tanaka, P. Wen, D. W. Kufe, W.G. Kaelin, Jr., and H. A. Fine. 1997, Nat Med. 3:1145–9.) Thus, weisolated the human E2F-1 promoter fragment from base pairs −218 to +51,relative to the transcriptional start site, by PCR with primers thatincorporated a SpeI and XhoI site into them. This creates the same sitespresent within the E4 promoter shuttle and allows for directsubstitution of the E4 promoter with the E2F-1 promoter. The details ofthe construction of this vector are described more in the Examples.

One embodiment of the invention is the description of an adenovirus E1aand/or E4 shuttle vector that allows fast and easy substitution of theendogenous nucleotide transcriptional regulatory sequences, where suchsequences are preferably E1a and/or E4 promoter sequences, withnucleotide transcriptional regulatory sequences that are response toelements (i.e. molecules) in the pRb signaling pathway, includingpRb/p107, E2F transcription factors such as E2F-1/-2/-3, and G1cyclin/cdk complexes. An E1a or E4 adenoviral vector, as describedabove, would be expected to be attenuated in normal cells that containan intact, that is wild type pRb pathway, yet exhibit a normal infectionprofile in cells that are deficient in Rb pathway function, includingfor pRb's repressive function. Due to the presence of the autoregulatoryE2F sites in the E2F-1 promoter, any E1A or E4 adenoviral vector havingnucleotide transcriptional regulatory sequences that are response toelements in the pRb signaling pathway subsituted for the endogenous E1aand/or E4 sequences will preferably have a second mutation in theE1A-CR2 (conserved region 2) domain. This is desirable to minimize E1A'sability to disrupt pRb-mediated repression of the E2F elements.

As referred to above, the adenoviral oncoprotein E1a, disrupts thepRB/E2F complex resulting in the release and thus the activation of E2F.The preferred E1a and/or E4 adenovirus shuttle vector construct is onethat is mutant in those regions of E1a that bind to pRb and displaceE2F. Thus, suitable E1a-RB replication deficient adenovirus constructsfor use in the methods and compositions of the invention to generate theinvention E1a and/or E4 shuttle vectors include, but are not limited tothe following examples: (1) adenovirus serotype 5 NT dl 1010, whichencodes an E1a protein lacking the CR1 and CR2 domains (deletion ofamino acids 2 to 150; nucleotides 560–1009) necessary for efficient RBbinding, but substantially retaining the CR3 domain (Whyte et al. (1989)Cell 56: 67), and (2) adenovirus serotype 5 dl 312, which comprises adeleted viral genome lacking the region spanning nucleotides 448–1349which encodes the entire E1a region in wild-type adenovirus (Jones N andShenk T (1979) Proc. Natl. Acad. Sci. (U.S.A.) 76: 3665). Ad5 NT dl 1010is a preferred E1a-RB replication deficient adenovirus and is availablefrom Dr. E. Harlow, Massachusetts General Hospital, Boston, Mass.).

Additional E1a mutants lacking the capacity to bind RB (E1a⁽⁻⁾) can begenerated by those of skill in the art by generating mutations in theE1a gene region encoding E1a polypeptides, typically in the CR1 and/orCR2 domains, expressing the mutant E1a polypeptide, contacting themutant E1a polypeptides with p105 or a binding fragment of RB underaqueous binding conditions, and identifying mutant E1a polypeptideswhich do not specifically bind RB as being candidate E1a⁽⁻⁾ mutantssuitable for use in the invention. Alternative assays include contactingthe mutant E1a polypeptides with the 300 kD protein and/or p107 proteinor binding fragment thereof under aqueous binding conditions, andidentifying mutant E1a polypeptides which do not specifically bind the300 kD and/or p107 polypeptides as being candidate E1a⁽⁻⁾mutantssuitable for use in the invention in the production of the E1a and/or E4shuttle vectors. Alternative binding assays include determining theinability of E1a⁽⁻⁾ mutant protein (or absence of E1a protein) to formcomplexes with the transcription factor E2F and/or to lack the abilityto dissociate the RB protein from RB:E2F complexes under physiologicalconditions (Chellappan et al. (1991) op.cit.).

Alternatively, functional assays for determining mutants lacking E1afunction, such as loss of transctivation by E1a of transcription ofvarious reporter polypeptides linked to a E1a-dependent transcriptionalregulatory sequence, and the like, will be used. Such inactivatingmutations typically occur in the E1a CR1 domain (amino acids 30–85 inAd5: nucleotide positions 697–790) and/or the CR2 domain (amino acids120–139 in Ad5; nucleotide positions 920–967), which are involved inbinding the p105 RB protein and the p107 protein. Preferably, the CR3domain (spanning amino acids 150–186) remains and is expressed as atruncated p289R polypeptide and is functional in transactivation ofadenoviral early genes.

It is important to note that while the E2F responsive promoter humanE2F-1 is the preferred promoter to replace the E1a and/or E4 endogenouspromoters that any E2F responsive nucleotide sequence that is activated,directly or indirectly, by elements in the pRb pathway will adequatelysubstitute for the endogenous promoters.

It is also important to note that while the construction of the E1aand/or E4 adenoviral vectors involves the removal of certaintranscriptional nucleotide start sites that the exact number of suchsites removed or retained should not be construed as limiting theinvention. What is intended in describing the invention is that in theplace of the endogenous promoters, the E2F responsive promoter functionsto drive the E1a and/or E4 genes to kill tumor cells. This process willvary in degree depending on the number or type of transcriptional startsites that are present in the E2F responsive promoter.

It is important to note that while the invention described herein ispresented in terms of adenovirus and an E2F responsive promoter, thatthe invention is not limited to adenovirus. Indeed, the skilledpractitioner of this art will recognize applications to virtually allviruses that exhibit a life cycle similar to adenovirus such that an E2Fresponsive promoter can be incorporated to control the expression ofcertain genes that confer on such viruses selective tumor cell killing.

USES OF THE INVENTION

As mentioned above, the invention adenoviruses can be used to treatdiseases which have altered pRb pathway function. Additionally,adenoviral therapy of the present invention may be combined with otherantineoplastic protocols, such as conventional chemotherapy, or withother viruses. See U.S. Pat. No. 5,677,178. Chemotherapy may beadministered by methods well known to the skilled practitioner,including systemically, direct injection into the cancer, or bylocalization at the site of the cancer by associating the desiredchemotherapeutic agent with an appropriate slow release material orintra-arterial perfusing the tumor. The preferred chemotherapeutic agentis cisplatin, and the preferred dose may be chosen by the practitionerbased on the nature of the cancer to be treated, and other factorsroutinely considered in administering cisplatin. Preferably, cisplatinwill be administered intravenously at a dose of 50–120 mg/m² over 3–6hours. More preferably it is administered intravenously at a dose of 80mg/m² over 4 hours. A second chemotherapeutic agent, which is preferablyadministered in combination with cisplatin is 5-fluorouracil. Thepreferred dose of 5-fluorouracil is 800–1200 mg/m² per day for 5consecutive days.

Adenoviral therapy using the instant invention adenoviruses may becombined with other antineoplastic protocols, such as gene therapy. See,U.S. Pat. No. 5,648,478. As mentioned above, adenovirus constructs foruse in the instant invention will exhibit specific cancer cell killing.Such constructs may also have prodrug activator genes, includingthymidine kinase, cytosine deaminase, or others, that in the presence ofthe appropriate prodrug will enchance the antineoplastic effect of theinvention E1a and/or E4 adenovirus vectors. See, U.S. Pat. No.5,631,236.

Also, in the event that the instant invention adenoviral mutants elicitan immune response that dampens their effect in a host animal, they canbe administered with an appropriate immunosuppressive drug to maximizetheir effect. Alternately, a variety of methods exist whereby theexterior protein coat of adenovirus can be modified to produce lessimmunogenic virus. See, PCT/US98/0503 where it is shown that a majorimmunogenic component of adenovirus' exterior coat, hexon protein, canbe genetically engineered to be less immunogenic. This is done bycreating a chimeric hexon protein by substituting for normal viral hexonprotein epitopes a sequence of amino acids not normally found in hexonprotein. Such adenoviral constructs are less immunogenic than the wildtype virus.

Another aspect of the instant invention is the incorporation ofheterologous genes into the E1a and/or E4 shuttle vectors, preferably inthe E1B, E3 regions of the virus. Examples of such heterologous genes,or fragments thereof that encode biologically active peptides, includethose that encode immunomodulatory proteins, and, as mentioned above,prodrug activators (i.e. cytosine deaminase, thymidine kinase, U.S. Pat.Nos. 5,358,866, and 5,677,178). Examples of the former would includeinterleukin 2, U.S. Pat. No. 4,738,927 or 5,641,665; interleukin 7, U.S.Pat. No. 4,965,195 or 5,328,988; and interleukin 12, U.S. Pat. No.5,457,038; tumor necrosis factor alpha, U.S. Pat. No. 4,677,063 or5,773,582; interferon gamma, U.S. Pat. No. 4,727,138 or 4,762,791; orGM-CSF, U.S. Pat. No. 5,393,870 or 5,391,485. Additionalimmunomodulatory proteins further include macrophage inflammatoryproteins, including MIP-3, (See, Well, T. N. and Peitsch, M C. J.Leukoc. Biol vol 61 (5): pages 545–50,1997), and cell suicide, orapoptosis inducing proteins, including BAD and BAX. See, Yang, E., etal. Cell, vol 80, pages 285–291 (1995); and Sandeep, R., et al Cell,vol. 91, pages 231–241 (1997). Monocyte chemotatic protein (MCP-3 alpha)may also be used. A preferred embodiment of a heterologous gene is achimeric gene consisting of a gene that encodes a protein that traverescell membranes, for example, VP22 or TAT, fused to a gene that encodes aprotein that is preferably toxic to cancer but not normal cells.

To increase the efficacy of the invention adenoviral E1A mutantconstructs they may be modified to exhibit enhanced tropism forparticular tumor cell types. For example, as shown in PCT/US98/04964 aprotein on the exterior coat of adenovirus may be modified to display achemical agent, preferably a polypeptide, that binds to a receptorpresent on tumor cells to a greater degree than normal cells. Also see,U.S. Pat. No. 5,770,442 and U.S. Pat. No. 5,712,136. The polypeptide canbe antibody, and preferably is single chain antibody.

Purification of Adenoviral Mutants

Adenovirus is routinely purified by a number of techniques includingcesium chloride banding using an ultracentrifuge. However, for largescale production of adenovirus, methods which give larger yields thanthose readily obtainable by cesium chloride ultracentrifugation aredesirable, and involve one or more chromatographic steps. The preferredmethod utilizes ion exchange chromatography. See, for example,PCT/US97/21504; and Huyghe et al., Human Gene Therapy, vol. 6: 1403–1416(1996).

FORMULATION

Adenovirus, including adenoviral mutants, may be formulated fortherapeutic and diagnostic administration to a patient. For therapeuticor prophylactic uses, a sterile composition containing apharmacologically effective dosage of adenovirus is administered to ahuman patient or veterinary non-human patient for treatment, forexample, of a neoplastic condition. Generally, the composition willcomprise about 10³ to 10¹⁵ or more adenovirus particles in an aqueoussuspension. A pharmaceutically acceptable carrier or excipient is oftenemployed in such sterile compositions. A variety of aqueous solutionscan be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine andthe like. These solutions are sterile and generally free of particulatematter other than the desired adenoviral vector. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents and the like, for example sodiumacetate, sodium chloride, potassium chloride, calcium chloride, sodiumlactate, etc. Excipients which enhance infection of cells by adenovirusmay be included.

Adenoviruses of the invention, or the DNA contained therein, may also bedelivered to neoplastic cells by liposome or immunoliposome delivery;such delivery may be selectively targeted to neoplastic cells on thebasis of a cell surface property present on the neoplastic cellpopulation (e.g., the presence of a cell surface protein which binds animmunoglobulin in an immunoliposome). Typically, an aqueous suspensioncontaining the virions are encapsulated in liposomes or immunoliposomes.For example, a suspension of adenovirus virions can be encapsulated inmicelles to form immunoliposomes by conventional methods (U.S. Pat. No.5,043,164, U.S. Pat. No. 4,957,735, U.S. Pat. No. 4,925,661; Connor andHuang (1985) J. Cell Biol. 101: 582; Lasic DD (1992) Nature 355: 279;Novel Drug Delivery (eds. Prescott L F and Nimmo W S: Wiley, New York,1989); Reddy et al. (1992) J. Immunol. 148: page 1585). Immunoliposomescomprising an antibody that binds specifically to a cancer cell antigen(e.g., CALLA, CEA) present on the cancer cells of the individual may beused to target virions, or virion DNA to those cells.

The compositions containing the present adenoviruses or cocktailsthereof can be administered for prophylactic and/or therapeutictreatments of neoplastic disease. In therapeutic application,compositions are administered to a patient already affected by theparticular neoplastic disease, in an amount sufficient to cure or atleast partially arrest the condition and its complications. An amountadequate to accomplish this is defined as a “therapeutically effectivedose” or “efficacious dose.” Amounts effective for this use will dependupon the severity of the condition, the general state of the patient,and the route of administration.

In prophylactic applications, compositions containing the inventionadenoviruses, or cocktails thereof, are administered to a patient notpresently in a neoplastic disease state to enhance the patient'sresistance to recurrence of a cancer or to prolong remission time. Suchan amount is defined to be a “prophylactically effective dose.” In thisuse, the precise amounts again depend upon the patient's state of healthand general level of immunity.

EXAMPLES Example 1

E2F-1E4 Adenoviral Vector Construction.

The recombinant plasmid pAd75–100(14) was obtained from Patrick Hearingand contains the Ad5 dl309 fragment from the EcoRI site at 75.9 mapunits (m.u.) to the right end of the viral genome at 100 m.u. (a BamHIlinker is located at 100 m.u.) in pBR322 between the EcoRI and BamHIsites. This EcoRI to BamHI fragment was directly subcloned into Litmus28 (New England Biolabs) to generate L28:p75–100.dl309. The wild typeAd5 E3 sequence that is missing in dl309 (Ad5 nucleotides 30,005 to30,750) was restored by replacing the NotI to NdeI fragment inL28:p75–100.dl309 with a wild type NotI to NdeI fragment (Ad5nucleotides 29,510–31,089) from pAd5-SN (described in U.S. patent Ser.No. 09/347,604 unpublished in-house vector). This plasmid was designatedL28:p75–100.wt. In order to generate the promoter shuttle, a slightlysmaller vector was generated to be the mutagenesis template. PlasmidpKSII+:p94–100 was constructed by directly subcloning the EcoRV to BamHIfragment (Ad5 nucleotides 33,758 to 33,939) from pAD75–100 into pKSII+.A XhoI site at nucleotide 35,577 and a SpeI site at nucleotide 35,816were created using the Stratagene Quickchange site directed mutagenesismethod. The oligonucleotides used to generate these sites were: XhoI(5′-GCTGGTGCCGTCTCGAGTGGTGTTTTTTTAATAGG-3′ (SEQ ID NO: 1) and itscomplement 5′-CCTATTAAAAAAACACCACTCGAGACGGCACCAGC-3′ (SEQ ID NO:2) andSpeI (5′-GGGCGGAGTAACTAGTATGTGTTGGG-3′ (SEQ ID NO:3) and its complement5′-CCCAACACATACTAGTTACTCCGCCC-3′ (SEQ ID NO:4). This vector containingboth the SpeI and XhoI restriction sites was designated pKSII+:E4PSV.Due to the presence of both a SpeI and XhoI site in the pKSII+ backbone,the EcoRV to BamHI fragment from pKSII+:E4PSV was subcloned into pRSET(Invitrogen) via the PvuII and BamHI sites and was designated aspRSET:E4PSV. All vectors and point mutations were verified by doublestranded sequence analysis on an ABI automated sequencer.

The human E2F-1 promoter was isolated by the polymerase chain reaction(PCR) from templates pGL3:E2F-1(−242) and pGL3:E2F-1ΔE2F(−242).pGL3:E2F-1(−242) contains a wild type human E2F-1 promoter out toposition −242 relative to the transcriptional start site.pGL3:E2F-1ΔE2F(−242) contains the same sequences except that both of theE2F binding-site palindromes contain inactivating point mutations. Theprimers used for PCR were as follows: SpeI-E2F1P(5′-GTGAGCACTAGTCGCCTGGTACCATCCGGACAAAGCC-3′SEQ ID NO:5) and XhoI-E2F1P(5′-GTGAGCCTCGAGCTCGATCCCGCTCCGCCCCCGG-3′SEQ ID NO:6). One hundrednanograms of template DNA were PCR amplified using Pfu DNA polymerase(Stratagene) under the following conditions: an initial denaturation at98° C. for 5 min., followed by 30 cycles of denaturation at 98° C. for 1min. and annealing/primer extension at 68° C. for 1 min., followed by afinal primer extension at 68° for 5 min. The PCR products were Qiagenpurified, digested with SpeI and XhoI, gel purified, and ligated intoSpeI and XhoI digested pRSET:E4PSV. These promoter shuttle vectors weredesignated E2F1-E4PSV and E2F1Δ-E4PSV and carry sequences from −218 to+51 relative to the transcriptional start of the human E2F1 promoter.The final vectors used to generate functional virus were created bysubcloning the BstEII to BamHI fragments from both E2F1-E4PSV andE2F1Δ-E4PSV into both L28:p75–100.dl309 and L28:p75–100.wt digested withsame enzymes. These vectors were designated as: E2F1-E4PSV.309,E2F1-E4PSV.wt, E2F1Δ-E4PSV.309, and E2F1Δ-E4PSV.wt. All vectors wereconfirmed by double stranded sequence analysis as described above.

Example 2

E2F1-E4 Adenovirus Construction.

Ten micro grams of E2F1-E4PSV.309 were digested with EcoRI and BamHI,treated with calf-intestinal phosphatase, and gel purified. One microgram of EcoRI digested dl922/47 TP-DNA was ligated to ˜5 micro grams ofthe purified fragment containing the wild type E2F-1 promoter drivingthe E4 region overnight at 16° C. Ligations were transfected into 293cells using standard a CaPO₄ transfection method. Briefly, the ligatedDNA was mixed with 24 micro grams of salmon sperm DNA, 50 micro litersof 2.5M CalCl₂, and adjusted to a final volume of 500 micro liters withH₂O. This solution was added dropwise to 500 micro liters ofHepes-buffered saline solution, pH 7.05. After standing for 25 minutes,the precipitate was added dropwise to two 60 mm dishes of 293 cellswhich had been grown in DMEM supplemented with 10% fetal bovine serum(FBS) to 60–80% confluency. After 16 hours, the monolayer was washed onetime with phosphate-buffered saline (minus calcium and magnesium)followed by a 5 ml agar overlay consisting of 1% Seaplaque agarose inDMEM supplemented with 2% FBS. Dishes were overlaid with 3–4 ml of theabove agar overlay every 3–4 days until plaques were isolated.

Example 3

E2F1-E4 Viral Propagation and Confirmation.

Primary plaques were isolated with a pasteur pipette and propagated in a6 well dish on 293 cells in 2 ml of DMEM supplemented with 2% FBS untilthe cytopathic effect (CPE) was complete. One-tenth (200 ml) of theviral supernatant was set aside for DNA analysis, while the remainderwas stored at −80° C. in a cryovial. DNA was isolated using Qiagen'sBlood Kit as per their recommendation. One-tenth of this material wasscreened by PCR for the presence of the desired mutations using thefollowing primers: for dl922/47(5′-GCTAGGATCCGAAGGGATTGACTTACTCACT-3′(SEQ ID NO: 7) and5′-GCTAGAATTCCTCTTCATCCTCGTCGTCACT-3′ SEQ ID NO:8) and for the E2F-1promoter in the E4 region (5′-GGTGACGTAGGTTTTAGGGC-3′ (SEQ ID NO:9) and5′-GCCATAACAGTCAGCCTTACC-3′ SEQ ID NO: 10). PCR was performed usingClontech's Advantage cDNA PCR kit in a Perkin Elmer 9600 machine usingthe following conditions: an initial denaturation at 98° C. for 5 min.,followed by 30 cycles of denaturation at 98° C. for 1 min. andannealing/primer extension at 68° C. for 3 min., followed by a finalprimer extension at 68° for 5 min. Positive plaques (as determined byPCR) were subsequently verified by sequence analysis. The above PCRproducts were gel purified and sequenced with the same primers. Positiveplaques were then subjected to a second round of plaque purification andverified as before. Viruses were propagated in 293 cells and purified bytwo rounds of cesium chloride gradient ultracentrifugation.

Example 4

E2F1-E1a and E2F1-E1a/E2F1-E4 Vector Construction.

The human E2F-1 promoter was isolated by the polymerase chain reaction(PCR) from templates pGL3:E2F-1(−242) and pGL3:E2F-1ΔE2F(−242).pGL3:E2F-1(−242) contains a wild type human E2F1 promoter out toposition −242 relative to the transcriptional start site.pGL3:E2F-1ΔE2F(−242) contains the same sequences except that both of theE2F binding-site palindromes contain inactivating point mutations. Theprimers used for PCR were as follows: BamHI-E2F1P(5′-GTGAGCGGATCCGCTCGATCCCGCTCCGCCCCCGG-3′ SEQ ID NO:11) andHindIII-E2F1P (5′-GTGAGCAAGCTTCGCCTGGTACCATCCGGACAAAGCC-3′ SEQ ID NO:12). One hundred nanograms of template DNA were PCR amplified using PfuDNA polymerase (Stratagene) under the following conditions: an initialdenaturation at 98° C. for 5 min., followed by 30 cycles of denaturationat 98° C. for 1 min. and annealing/primer extension at 68° C. for 1min., followed by a final primer extension at 68° for 5 min. The PCRproducts were purified over Qiaquick columns (Qiagen), digested withBamHI and HindIII, gel purified, and ligated into BamHI and HindIIIpartially digested p922/47-SV (see below). These promoter shuttlevectors were designated E2F1wt-922/47.PSV and E2F1Δ-922/47.PSV and carrysequences from −218 to +51 relative to the transcriptional start of thehuman E2F1 promoter. All vectors were confirmed by double strandedsequence analysis on an ABI automated sequencer.

P922/4′-SV is an E1A promoter shuttle vector that also contains anE1A-CR2 deletion from nucleotides 922 to 947. Plasmid P922/47-SV wasconstructed by first digesting pSP64 (Promega) with HindIII, bluntingwith Klenow DNA polymerase, and then religating to generate pSP64 DeltaH3. The 1,737 bp EcoRI to XbaI fragment (containing both Ad5 and pBR322DNA) from pXC1 (Microbix) was then ligated into EcoRI and XbaI digestedpSP64 Delta H3 to generate pSP64-RI/Xba. pSP64-RI/Xba was then digestedwith HindIII and BamHI, blunted with Klenow DNA polymerase and religatedto generate P Delta E1 Delta +. This intramolecular deletion removedsequences from 9529 to 9875 of the pXC1 plasmid, effectively removingthe HindIII, BamHI and ClaI sites. A novel HindIII site at nucleotide376 of Ad5 was then created by digesting P Delta E1 Delta with BsAaI andligating in a HindIII linker (NEB) to generate P Delta E1 Delta +H. Anovel BamHI site was then created at nucleotide 539 of Ad5 by PCRmutagenesis. Two initial PCR reactions were performed. P Delta E1 Delta+H was used as a template with a primer 5′EcoXC1 site present in pBR322and 3′ Bam (5′-CGCGGAATTCTTTTGGATTGAAGCCAATATG-3′ SEQ ID NO:13) and 3′Bam (5′-CAGTCCCGGTGTCGGATCCGCTCGGAGGAG-3′ SEQ ID NO: 14), whereasplasmid pXC1 (Microbix) was used as the template in a PCR reaction withprimers Bsr-Bam (5′-CTCCTCCGAGCGGATCCGACACCGGGACTG-3′ SEQ ID NO: 15) and3′ E1A.Xba (5′-GCGGGACCACCGGGTGTATCTCAGGAGGTG-3′ SEQ ID NO:16). The PCRproducts were isolated on an agarose gel and purified using a Qiagen gelextraction kit. The two PCR products were then mixed and PCR wasrepeated using the external most primers 5′EcoXC1 and 3′E1A.Xba. Theresulting ˜1,400 bp PCR product was then digested with EcoRI and XbaIand ligated into EcoRI and XbaI digested P Delta E1 Delta +H to generateDelta E1 Delta +H+B. pXC1-SV was then constructed by digesting P DeltaE1 Delta +H+B with EcoRI and XbaI and ligating the 1,393 bp fragmentinto EcoRI and XbaI digested pXC1 (Microbix). Finally, p922/47-SV wasgenerated by using pCIA-922/47 (provided by Peter White) as a templatefor PCR with the following primers: Bsr-Bam(5′-CTCCTCCGAGCGGATCCGACACCGGGACTG-3′ SEQ ID NO:15) and 3′E1A.Xba(5′-GCATTCTCTAGACACAGGTG-3′ SEQ ID NO:17). The resulting PCR product waspurified over a Qiagen Qiaquick column, digested with BamHI and XbaI andsubsequently ligated into pXC1-SV that had been digested with BamHI andXbaI.

Example 5

E2F1-E1a and E2F1-E1a/E2F1-E4Viral Construction.

ONYX-150 (E2F1wt-922/47) and ONYX-151 (E2F1 Delta-922/47) were generatedby cotransfecting 10 micro grams of either E2F1wt-922/47.PSV or E2F1Delta-922/47.PSV, respectively, with 10 micro grams of pJM17 (Microbix)into 293 cells using a standard CaPO₄ transfection method. ONYX-411(E2F1wt-922/47+E2F1wt-E4) was generated by digesting 10 micro grams ofplasmid E2F1-E4PSV.309 (ID-086) with EcoRI and BamHI. The digested DNAwas then treated with calf-intestinal phosphatase and gel purified. Onemicrogram of EcoRI digested ONYX-150 (E2F1wt-922/47) TP-DNA was thenligated to 5 micro grams of the purified fragment containing the wildtype E2F-1 promoter driving the E4 region overnight at 16° C. CaPO₄transfections were performed by mixing the DNA's with 50 micro liters of2.5 M CaCl₂, in a final volume of 500 micro liters. In the case ofONYX-411, the transfection mix contained 24 micrograms of salmon spermDNA in addition to the ligated DNA's. This solution was added dropwiseto 500 micro liters of Hepes-buffered saline solution, pH 7.05. Afterstanding for 25 minutes, the precipitate was added dropwise to two 60 mmdishes of 293 cells which had been grown in DMEM supplemented with 10%fetal bovine serum (FBS) to 60–80% confluency. After 16 hours, themonolayer was washed one time with phosphate-buffered saline (minuscalcium and magnesium) followed by a 5 ml agar overlay consisting of 1%Seaplaque agarose in DMEM supplemented with 2% FBS. Dishes were overlaidwith 3–4 ml of the above agar overlay every 3–4 days until plaques wereisolated.

Example 6

E2F1-E1a and E2F1-E1a/E2F1-E4 Viral Propagation and Confirmation.

Primary plaques were isolated with a pasteur pipette and propagated in a6 well dish on either 293 or A549 cells in 2 ml of DMEM supplementedwith 2% FBS until the cytopathic effect (CPE) was complete. One-tenth(200 micro liters) of the viral supernatant was set aside for DNAanalysis, while the remainder was stored at −80° C. in a cryovial. DNAwas isolated using Qiagen's Blood Kit as per their recommendation.One-tenth of this material was screened by PCR for the presence of thedesired mutations using the following sets of primer pairs. The presenceof the human E2F1 promoter driving E1A was confirmed using primersAd5-left (5′-GGGCGTAACCGAGTAAGATTTGGCC-3′ SEQ ID NO: 18) and E1Astart.NC(5′-GGCAGATAATATGTCTCATTTTCAGTCCCGG-3′ SEQ ID NO: 19). The presence ofthe deletion from nucleotides 922 to 947 within E1A was verified usingprimers Af-7 (5′-GCTAGGATCCGAAGGGATTGACTTACTCACT-3′ SEQ ID NO:20) andAf-5 (5′-GCTAGAATTCCTCTTCATCCTCGTCGTCACT-3′ SEQ ID NO:21). The presenceof the human E2F1 promoter driving the entire E4 region was confirmedusing primers E4.3NCb (5′-GCCATAACAGTCAGCCTTACC-3′ SEQ ID NO:22) andAd5-3′ end (5′-GGTGACGTAGGTTTTAGGGC-3′ SEQ ID NO:23). The deletionpresent in the E3 region (dl309) was confirmed using primers E3.C8(5′-CCTTTATCCAGTGCATTGACTGGG-3′ SEQ ID NO.:24) and 3′-E3I(5′-GGAGAAAGTTTGCAGCCAGG-3′ SEQ ID NO:25). PCR was performed usingClontech's Advantage cDNA PCR kit in a Perkin Elmer 9600 machine usingthe following conditions: an initial denaturation at 98° C. for 5 min.,followed by 30 cycles of denaturation at 98° C. for 1 min. andannealing/primer extension at 68° C. for 3 min., followed by a finalprimer extension at 68° for 5 min. Positive plaques (as determined byPCR analysis) were subsequently verified by sequence analysis. The abovePCR products were gel purified and sequenced with the same primers.Positive plaques were then subjected to a second round of plaquepurification in either 293 or A549 cells and verified exactly as before.Viruses were propagated in 293 cells and purified by two rounds ofcesium chloride gradient ultracentrifugation. All large-scale viralpreps were confirmed by the above same PCR and sequence analyses. Inaddition, all large-scale viral preps were verified by digestion witheither HindIII or XhoI and the fragments analyzed by isolation on a 0.9%agarose gel.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the appendedclaims.

1. An adenoviral vector comprising E2F binding sites that control theexpression of an early adenoviral gene, and a mutation in the E1a regionof said adenoviral vector, which mutation causes a loss of RB binding tothe protein encoded by the E1a region.
 2. An adenoviral vector asdescribed in claim 1, wherein said E2F binding sites comprise an E2Fpromoter.
 3. An adenoviral vector as described in claim 2, wherein saidE2F binding sites are substituted for an endogenous adenoviral E1apromoter.
 4. An adenoviral vector as described in claim 3, wherein saidadenoviral vector further comprises nucleotide regulatory sites thatfacilitate adenoviral replication comprising Sp1, ATF, NF1 andNFIII/Oct-1 binding sites.
 5. An adenoviral vector comprising a viraltranscriptional nucleotide regulatory site that controls the expressionof an early adenoviral gene, wherein said site is inactivated by theinsertion of E2F binding sites such that said E2F binding sites controlthe expression of said adenoviral gene, and said adenoviral vectorfurther comprises a mutation in the E1a region of, which mutation causesa loss of RB binding to the protein encoded by the E1a region.
 6. Anadenoviral vector as described in claim 5, wherein said adenoviralinactivated transcriptional nucleotide regulatory site is a promoter. 7.An adenoviral vector as described in claim 6 wherein said inactivatedtranscriptional nucleotide regulatory site is an endogenous adenoviralE1a promoter.
 8. An adenoviral vector as described in claim 6, whereinsaid inactivated transcriptional nucleotide regulatory site comprisesboth an endogenous adenoviral E1a and E4 promoters.
 9. An adenoviralvector as described in claim 5, wherein said E2F binding sites, comprisean E2F promoter.
 10. An adenoviral vector as described in claim 2 or 9,wherein said E2F promoter is a human E2F-1 promoter.
 11. A method forkilling cancer cells in the presence of normal cells, comprising thesteps of: contacting under infective conditions an adenoviral vector asdescribed in claim 1 or 5 with a cell population comprising cancer andnormal cells, and allowing sufficient time for said adenovirus to infectsaid cell population.